New stabilized FastPrep-CLEAs for sialic acid synthesis

N-acetyl-d-neuraminic acid aldolase, a key enzyme in the biotechnological production of N-acetyl-d-neuraminic acid (sialic acid) from N-acetyl-d-mannosamine and pyruvate, was immobilized as cross-linked enzyme aggregates (CLEAs) by precipitation with 90% ammonium sulfate and crosslinking with 1% glutaraldehyde. Because dispersion in a reciprocating disruptor (FastPrep) was only able to recover 40% of the activity, improved CLEAs were then prepared by co-aggregation of the enzyme with 10 mg/mL bovine serum albumin followed by a sodium borohydride treatment and final disruption by FastPrep (FastPrep-CLEAs). This produced a twofold increase in activity up to 86%, which is a 30% more than that reported for this aldolase in cross-linked inclusion bodies (CLIBs). In addition, these FastPrep-CLEAs presented remarkable biotechnological features for Neu5Ac synthesis, including, good activity and stability at alkaline pHs, a high K_M for ManNAc (lower for pyruvate) and good operational stability. These results reinforce the practicability of using FastPrep-CLEAs in biocatalysis, thus reducing production costs and favoring reusability.     

The synthesis and evaluation of novel sialic acid analogues bound to matrices for the purification of sialic acid-recognising proteins

A novel N-acetylneuraminic acid analogue, 2-S-(5'-aminopentyl) 5-acetamido-3,5-dideoxy-2-thio-D-glycero-alpha-D-galacto-2- nonulopyranosidonic acid, as well as the thiosialoside 2-S-(2'-aminoethyl) 5-acetamido-3,5-dideoxy-2-thio-D-glycero-alpha-D-galacto-2- nonulopyranosidonic acid, have been synthesised and successfully coupled to CNBr-activated Sepharose 4B through the terminal amino group. The resultant affinity resins have proved efficient in purifying a number of sialic acid-recognising proteins such as Vibrio cholerae sialidase, sialidase-L from leech, trans-sialidase from Trypanosoma cruzi, and sialyltransferases from rat liver, all in high yield.     

Smart process development: Application of machine-learning and integrated process modeling for inclusion body purification processes

The development of a biopharmaceutical production process usually occurs sequentially, and tedious optimization of each individual unit operation is very time-consuming. Here, the conditions established as optimal for one-step serve as input for the following step. Yet, this strategy does not consider potential interactions between a priori distant process steps and therefore cannot guarantee for optimal overall process performance. To overcome these limitations, we established a smart approach to develop and utilize integrated process models using machine learning techniques and genetic algorithms. We evaluated the application of the data-driven models to explore potential efficiency increases and compared them to a conventional development approach for one of our development products. First, we developed a data-driven integrated process model using gradient boosting machines and Gaussian processes as machine learning techniques and a genetic algorithm as recommendation engine for two downstream unit operations, namely solubilization and refolding. Through projection of the results into our large-scale facility, we predicted a twofold increase in productivity. Second, we extended the model to a three-step model by including the capture chromatography. Here, depending on the selected baseline-process chosen for comparison, we obtained between 50% and 100% increase in productivity. These data show the successful application of machine learning techniques and optimization algorithms for downstream process development. Finally, our results highlight the importance of considering integrated process models for the whole process chain, including all unit operations.     

A ribonucleoprotein inclusion body in Entamoeba invadens

Summary Cytochemical tests have shown that the chromatoid bodies contain mainly ribonucleic acid and protein. There is no evidence to suggest the presence of any desoxyribonucleic acid, lipid, glycogen, neutral fat, or metaphosphate. The cyclic changes in the chromatoid body have been studied by means of light and electron microscopy and estimations of the total RNA present. The results indicate that the chromatoid bodies arise by a process of aggregation of small groups of 250–300 A units form polycrystalline masses in precystic and early cysts. At the same time there is a steady rise in the amount of RNA present. In the maturing cysts, the crystalline masses fragment into separate particles, but there is no corresponding fall in the amount of RNA. The significance of these results in relation to the previous literature on chromatoid bodies and similarly named cellular inclusions in spermatogenic and plant cells, as well as in the protozoa, is discussed. Attention is drawn to the striking similarity of composition, formation, development and ultimate fate of these various inclusion bodies but as the terminology is still confused it is proposed that the term chromatoid body be retained solely for particulate or lamellar ribonucleo-protein accumulations. Acknowledgements. Most of this study was presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the University of Edinburgh. I should like to express my deep appreciation to Prof. M. M. Swann F. R. S. for his interest and helpful guidance in this work. I am grateful to the Melville Trust for Cancer Research who provided the electron microscope.     

Differential expression of sialic acid on porcine organs during the maturation process

Sialylated structures play important roles in cell communication, and change in a regulated manner during development and differentiation. In this work, we report the main glycosidic modifications that occur during the maturation of porcine tissues, involving the sialylation process as determined with lectins. Sialic acids were identified at several levels in a broad range of cell types of nervous, respiratory, genitourinary and lymphoid origin. Nevertheless, the most contrasting was the type of glycosidic linkage between 5-N-acetyl-neuraminic acid (Neu5Ac) and galactose (Gal) expressed in central nervous system (CNS). Newborn CNS abundantly expressed Neu5Acalpha2,3Gal, but weakly or scarcely expressed Neu5Acalpha2,6Gal/GalNAc. Maturation of CNS induced drastic changes in sialic acid expression. These changes include decrease or complete loss of NeuAcalpha2,3Gal residues, mainly in olfactory structures and brain cortex, which were replaced by their isomers Neu5Acalpha2,6Gal/GalNAc. In the brain cortex and cerebellum, the increase of Neu5Acalpha2,6Gal/GalNAc molecules was paralleled by an increase of 5-N-acetyl-9-O-acetyl-neuraminic acid (Neu5,9Ac2). In addition, terminal Gal and N-acetyl-D-galactosamine (GalNAc) residues also increased their expression in adult CNS tissues, but this was more significant in structures forming the encephalic trunk. Our results show that sialylation of porcine CNS is finely modulated throughout the maturation process.     

Physiology of sialic acid capsular polysaccharide synthesis in serogroup B Neisseria meningitidis

The pathway for biosynthesis of sialic acid capsular polysaccharide was examined in Neisseria meningitidis serogroup B strain M986 and in strain PRM102, an isogenic mutant defective in polysaccharide production. Strain PRM102 was found to possess only 25% of the level of sialyltransferase activity that was found in strain M986, but it had wild-type levels of both the N-acetylneuraminic acid (NANA) condensing enzyme and the CMP-NANA synthetase. A new meningococcal enzyme, a CMP-NANA hydrolase, was found in both meningococcal strains. This enzyme generated CMP and NANA from CMP-NANA, had a Km of 0.88 microM, had a Vmax of 10.75 nmol of NANA produced per h per mg of protein, and was completely inhibited by 45.3 microM CMP. The sialyltransferase, which also had CMP-NANA as substrate, was insensitive to CMP addition. Subcellular fractionation and purification of cytoplasmic and outer membranes on sucrose density gradients revealed that both the sialyltransferase and the CMP-NANA hydrolase were cytoplasmic membrane associated. The NANA condensing enzyme and the CMP-NANA synthetase were found to be cytosolic. A working hypothesis for the regulation of sialic acid polysaccharide synthesis was developed. The CMP-NANA hydrolase with its high affinity for CMP-NANA regulates polysaccharide formation by the sialyltransferase, whereas CMP, a product of both the sialyltransferase and the CMP-NANA hydrolase, modulates the activity of the hydrolase on the cytoplasmic membrane.     

Enzymatic synthesis of sialic acid derivative by immobilized lipase from Candida antarctica

The effects of important reaction parameters on the enhancement of sialic acid derivative lipophilic properties through the lipase-catalyzed esterification of N-acetyl neuraminic acid methyl ester are investigated in this study. It is found that the lipase Novozym 435 from Candida antarctica is particularly useful in the preparation of sialic acid methyl ester monononanoate (SAMEMN). The optimum temperature for the SAMEMN synthesis reaction using Novozym 435 is 60 °C, and nonanoic anhydride is found to be the best substrate among all acyl donors. The Novozym 435-catalyzed esterification of N-acetyl neuraminic acid methyl ester gave a maximum yield of 87.7% after 6 h in acetonitrile at 60 °C. Because the novel method developed is simple, yet effective, it could potentially be used industrially for the production of sialic acid derivatives.     

Parallel chemoenzymatic synthesis of sialosides containing a C5-diversified sialic acid

A convenient chemoenzymatic strategy for synthesizing sialosides containing a C5-diversified sialic acid was developed. The α2,3- and α2,6-linked sialosides containing a 5-azido neuraminic acid synthesized by a highly efficient one-pot three-enzyme approach were converted to C5″-amino sialosides, which were used as common intermediates for chemical parallel synthesis to quickly generate a series of sialosides containing various sialic acid forms.     

The impact of technical failures during cultivation of an inclusion body process

Bioprocess and Biosystems Engineering - In biotechnological processes, technical failures in the upstream process often lead to batch loss. It is of great interest to investigate the empirical...     

Techno-economic evaluation of an inclusion body solubilization and recombinant protein refolding process

Expression of recombinant proteins in Escherichia coli is normally accompanied by the formation of inclusion bodies (IBs). To obtain the protein product in an active (native) soluble form, the IBs must be first solubilized, and thereafter, the soluble, often denatured and reduced protein must be refolded. Several technically feasible alternatives to conduct IBs solubilization and on-column refolding have been proposed in recent years. However, rarely these on-column refolding alternatives have been evaluated from an economical point of view, questioning the feasibility of their implementation at a preparative scale. The presented study assesses the economic performance of four distinct process alternatives that include pH induced IBs solubilization and protein refolding (pH_IndSR); IBs solubilization using urea, dithiothreitol (DTT), and alkaline pH followed by batch size-exclusion protein refolding; inclusion bodies (IBs) solubilization using urea, DTT, and alkaline pH followed by simulated moving bed (SMB) size-exclusion protein refolding, and IBs solubilization using urea, DTT and alkaline pH followed by batch dilution protein refolding. The economic performance was judged on the basis of the direct fixed capital, and the production cost per unit of product (P(C)). This work shows that (1) pH_IndSR system is a relatively economical process, because of the low IBs solubilization cost; (2) substituting β-mercaptoethanol for dithiothreithol is an attractive alternative, as it significantly decreases the product cost contribution from the IBs solubilization; and (3) protein refolding by size-exclusion chromatography becomes economically attractive by changing the mode of operation of the chromatographic reactor from batch to continuous using SMB technology.     

Flexible crosslinked benzhydryl support for gel-phase peptide synthesis

An amphiphilic support was developed by the suspension polymerization of styrene and 1,4-butanediol dimethacrylate. The copolymer was converted to benzhydryl resin, BDDMA-PS-BH, by a two-step polymer analogous reaction. This resin was employed as a support for the synthesis of delta sleep inducing peptide (DSIP), Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu, in high yield and purity by Fmoc / t-Bu tactics.     

In situ proteolytic digestion of inclusion body polypeptides occurs as a cascade process

Misfolded proteins undergo a preferent degradation ruled by the housekeeping bacterial proteolytic system, but upon precipitation as inclusion bodies their stability dramatically increases. The susceptibility of aggregated polypeptides to proteolytic attack remains essentially unexplored in bacteria and also in eukaryotic cells. We have studied here the in vitro proteolysis of beta-galactosidase fusion proteins by trypsin treatment of purified inclusion bodies. A cascade digestion process similar to that occurring in vivo has been observed in the insoluble fraction of the digestion reaction. This suggests that major protease target sites are not either lost or newly generated by protein precipitation and that the digestion occurs in situ probably on solvent-exposed surfaces of inclusion bodies. In addition, the sequence of the proteolytic attack is influenced by protein determinants other than amino acid sequence, the early digestion steps having a dramatic influence on the further cleavage susceptibility of the intermediate degradation fragments. These observations indicate unexpected conformational changes of inclusion body proteins during their site-limited digestion, that could promote protein release from aggregates, thus partially accounting for the plasticity of in vivo protein precipitation and solubilization in bacteria.     

A second uniquely human mutation affecting sialic acid biology

Siglecs are immunoglobulin superfamily member lectins that selectively recognize different types and linkages of sialic acids, which are major components of cell surface and secreted glycoconjugates. We report here a human Siglec-like molecule (Siglec-L1) that lacks a conserved arginine residue known to be essential for optimal sialic acid recognition by previously known Siglecs. Loss of the arginine from an ancestral molecule was caused by a single nucleotide substitution that occurred after the common ancestor of humans with the great apes but before the origin of modern humans. The chimpanzee Siglec-L1 ortholog remains fully functional and preferentially recognizes N-glycolylneuraminic acid, which is a common sialic acid in great apes and other mammals. Reintroducing the ancestral arginine into the human molecule regenerates the same properties. Thus, the single base pair mutation that replaced the arginine on human Siglec-L1 is likely to be evolutionarily related to the previously reported loss of N-glycolylneuraminic acid expression in the human lineage. Siglec-L1 and its chimpanzee Siglec ortholog also have a different expression pattern from previously reported Siglecs because they are found on the lumenal edge of epithelial cell surfaces. Notably, the human genome contains several Siglec-like pseudogenes that have independent mutations that would have replaced the arginine residue required for optimal sialic acid recognition. Thus, additional changes in the biology of sialic acids may have taken place during human evolution.     

Investigation into an efficient synthesis of 2,3-dehydro-N-acetyl neuraminic acid leads to three decarboxylated sialic acid dimers

Sialic acid, an important carbohydrate found incorporated on the cell surface of many organisms, has been modified for use in a wide range of biological and pharmaceutical applications. We hypothesized that 4,7,8,9-tetra-O-acetyl-2-deoxy-2,3-dehydro-N-acetyl neuraminic acid methyl ester (4) could be efficiently synthesized in a one-pot reaction by heating peracetylated sialic acid (2) in pyridine and acetic anhydride to induce beta-elimination. When reduced to practice, this reaction produced only modest yields of 4. Six compounds, including three new decarboxylated sialic acid dimers, were also found to have been synthesized in the reaction. In an effort to better understand the chemistry and the mechanisms of this reaction, all of the side products were isolated and fully characterized.     

Molecular dynamics simulation studies on influenza A virus H5N1 complexed with sialic acid and fluorinated sialic acid

Abbreviations HA Hemagglutinin

MD Molecular Dynamics

MM-PBSA Molecular Mechanics Poisson–Boltzmann Surface Area

NA Neuraminidase

NAMD Nanoscale Molecular Dynamic Simulation

PMEMD Particle Mesh Ewald Molecular Dynamics

RMSD Root-Mean-Square Deviation

RMSF Root-Mean-Square Fluctuation

SIA sialic acid

VMD Visual Molecular Dynamics

Communicated by Ramaswamy H. Sarma     

A fibrous inclusion body in developing ascospores ofAscobolus stercorarius

Summary Electron microscopic examination, using glutaraldehyde and osmium tetroxide as fixatives, has revealed the existence of a unique structure, the filamentous ascospore inclusion (FAI), in the sporoplasm of developing ascospores ofAscobolus stercorarius. The FAI, a highly ordered aggregate of 80–125 å diameter, rigid, filamentous tubules, is examined as to structure and origin; in particular, certain evidence suggesting these structures are the aggregated form of the intra-nuclear spindle is discussed. The functional role of the FAI is considered in relation to the current literature; a bifunctional role in the apothecium is considered in light of the occurrence of an additional class of filamentous structure in other differentiated elements of the ascocarp.     

Versatile biosynthetic engineering of sialic acid in living cells using synthetic sialic acid analogues.

Sialic acids are critical components of many glycoconjugates involved in biologically important ligand-receptor interactions. Quantitative and structural variations of sialic acid residues can profoundly affect specific cell-cell, pathogen-cell, or drug-cell interactions, but manipulation of sialic acids in mammalian cells has been technically limited. We describe the finding of a previously unrecognized and efficient uptake and incorporation of sialic acid analogues in mammalian cells. We added 16 synthetic sialic acid analogues carrying distinct C-1, C-5, or C-9 substitutions individually to cell cultures of which 10 were readily taken up and incorporated. Uptake of C-5- and C-9-substituted sialic acids resulted in the structural modification of up to 95% of sialic acids on the cell surface. Functionally, binding of murine sialic acid-binding immunoglobulin-like lectin-2 (Siglec-2, CD22) to cells increased after N-glycolylneuraminic acid treatment, whereas 9-iodo-N-acetylneuraminic acid abolished binding. Furthermore, susceptibility to infection by the B-lymphotropic papovavirus via a sialylated receptor was markedly enhanced following pretreatment of host cells with selected sialic acid analogues including 9-iodo-N-acetylneuraminic acid. This novel experimental strategy allows for an efficient biosynthetic engineering of surface sialylation in living cells. It is versatile, extending the repertoire of modification sites at least to C-9 and enables detailed structure-function studies of sialic acid-dependent ligand-receptor interactions in their native context.